SEA-PHAGES | Frameshift in BM cluster phages?

Link to this post \| posted 16 Jun, 2022 15:45
kcornely	Greetings, all! We are annotating Frankenweenie, a Streptomyces BM cluster phage. We haven't annotated a BM cluster phage before. We were wondering if gp65 and gp66 were tail assembly chaperones and if gp66 was translated as a frameshift. There are six BM cluster phages, and the only phage in which the frameshift is called is Satis. It is not called for the other BM cluster phages. A frameshift seems likely for Frankenweenie because there is considerable overlap between gp65 and gp66. Thanks in advance for your help! Kathleen

Link to this post | posted 16 Jun, 2022 15:45

Greetings, all! We are annotating Frankenweenie, a Streptomyces BM cluster phage. We haven't annotated a BM cluster phage before. We were wondering if gp65 and gp66 were tail assembly chaperones and if gp66 was translated as a frameshift. There are six BM cluster phages, and the only phage in which the frameshift is called is Satis. It is not called for the other BM cluster phages. A frameshift seems likely for Frankenweenie because there is considerable overlap between gp65 and gp66. Thanks in advance for your help!
Kathleen

Link to this post \| posted 16 Jun, 2022 17:14
cdshaffer	We annotated Satis and using the rules that existed at the time we did find and annotate a slippery sequence, however looking at it now I am not sure that annotation is correct. Also when we did the second BM that slippery sequence we annotated was not conserved so at that point we were not convinced we have found a slippery sequence. So unless you have a really good match to an already documented slippery sequence (see the table here: bioinformatics guide "Annotating programmed translational frameshifts") I would not call the slip. It is still possible that you could assign the functional annotation of "tail assembly chaperone" to one or both of the two genes, irrespective of finding the slippery site. For that just use standard functional evidence of HHPRED, blast, synteny.

Link to this post | posted 16 Jun, 2022 17:14

cdshaffer

We annotated Satis and using the rules that existed at the time we did find and annotate a slippery sequence, however looking at it now I am not sure that annotation is correct. Also when we did the second BM that slippery sequence we annotated was not conserved so at that point we were not convinced we have found a slippery sequence. So unless you have a really good match to an already documented slippery sequence (see the table here: bioinformatics guide "Annotating programmed translational frameshifts") I would not call the slip. It is still possible that you could assign the functional annotation of "tail assembly chaperone" to one or both of the two genes, irrespective of finding the slippery site. For that just use standard functional evidence of HHPRED, blast, synteny.

Link to this post \| posted 16 Jun, 2022 17:52
debbie	I just looked at the tail assembly chaperones of Cluster BM. Here is my take: currently there are six members: Satis Kradla EhyElimyoE JustBecause Kela Frankenweenie To the best of my knowledge none of the protein sequences in question here show an HHPred hit to a tail assembly chaperone (TAC). In order to call them TACs, if they possessed a canonical slippage, they could be called TACs and the slippage would be annotated. The ribosomal slippage found in Satis looks great, AND is identical to what is in Kradal and EhyElimyoE (though they are not called….. yet). Neither JustBecause or Kela have that same slippage. And Frankenweenie doesn't look at bit like the others in the c-terminus. (do a blastp - sequence alignment with Satis_58 and you will see how the alignment falls apart. However, the sequence homology with Satis is compelling. i would be inclined to call both genes TACs, but do not slip them together. I think that is what I would do. Good luck! debbie

Link to this post | posted 16 Jun, 2022 17:52

debbie

I just looked at the tail assembly chaperones of Cluster BM.
Here is my take:
currently there are six members:
Satis
Kradla
EhyElimyoE
JustBecause
Kela
Frankenweenie

To the best of my knowledge none of the protein sequences in question here show an HHPred hit to a tail assembly chaperone (TAC). In order to call them TACs, if they possessed a canonical slippage, they could be called TACs and the slippage would be annotated.

The ribosomal slippage found in Satis looks great, AND is identical to what is in Kradal and EhyElimyoE (though they are not called….. yet).

Neither JustBecause or Kela have that same slippage.

And Frankenweenie doesn't look at bit like the others in the c-terminus. (do a blastp - sequence alignment with Satis_58 and you will see how the alignment falls apart.

However, the sequence homology with Satis is compelling. i would be inclined to call both genes TACs, but do not slip them together.

I think that is what I would do.
Good luck!
debbie

Link to this post \| posted 16 Jun, 2022 18:05
debbie	Kathleen and Chris, After reading Chris' entry, he is right -the slippage called in Satis, though it looks very slippery, is not canonical, i.e. not found in the literature. To that end, if we indeed are not calling any of the slippages, I would be inclined to NOT assign the function of a tail assembly chaperone (because there is no HHPred evidence of a TAC) unless I overlooked something.

Link to this post \| posted 17 Jun, 2022 02:00
kcornely	Thanks Chris and Debbie! This is incredibly helpful.

Link to this post \| posted 17 Jun, 2022 19:07
cdshaffer	The upstream "G" form of this gene has a weak hit to the Tail assembly chaperone Pfam. . PF16459.8. The numbers on this hit are pretty bad 68% probability. So by itself it certainly not sufficient evidence to call a TAC. However we thought by adding in synteny evidence and that we had found a "pretty good" possible slippery sequence that the sum of all those observations that the TAC function was justified. But I agree with the recent discussions elsewhere that there should be a policy which is to only call the slip if there is a sequence match to one of the published, well documented slippery sequences. So calling the slip here is an overcall. If you remove the slip then there is really no evidence for functionally calling the downstream "T" region as a TAC (except for synteny evidence), and while we do call some gene functions based only on synteny, TAC is not one of them. So if we want to update the annotation in Genbank for this phage, I would say keep the TAC function call for the G form (32294-33103), {based on weak hhpred evidence combined with good synteny evidence), remove the gene that codes for the GT form (32294..33055,33057..33407), and add in a gene that starts at 33072 or 33126 as a gene, and give it an NKF annotation. I would probably go with the longer start without evidence to the contrary but since there is a very high level of uncertainty here in any case I could go with either start. 93Kb

Link to this post | posted 17 Jun, 2022 19:07

cdshaffer

The upstream "G" form of this gene has a weak hit to the Tail assembly chaperone Pfam. . PF16459.8. The numbers on this hit are pretty bad 68% probability. So by itself it certainly not sufficient evidence to call a TAC. However we thought by adding in synteny evidence and that we had found a "pretty good" possible slippery sequence that the sum of all those observations that the TAC function was justified. But I agree with the recent discussions elsewhere that there should be a policy which is to only call the slip if there is a sequence match to one of the published, well documented slippery sequences. So calling the slip here is an overcall.

If you remove the slip then there is really no evidence for functionally calling the downstream "T" region as a TAC (except for synteny evidence), and while we do call some gene functions based only on synteny, TAC is not one of them. So if we want to update the annotation in Genbank for this phage, I would say keep the TAC function call for the G form (32294-33103), {based on weak hhpred evidence combined with good synteny evidence), remove the gene that codes for the GT form (32294..33055,33057..33407), and add in a gene that starts at 33072 or 33126 as a gene, and give it an NKF annotation. I would probably go with the longer start without evidence to the contrary but since there is a very high level of uncertainty here in any case I could go with either start.

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Frameshift in BM cluster phages?